| Citrus bacterial canker disease (CBCD), caused by Xanthomonas axonopodis pv. citri Vauterin et al. (Synonym Xanthomonas campestris pv. citri (Hasse) Dye, Xac), is a serious quarantine disease of most citrus species and cultivars in many citrus-producing areas worldwide. Wind-driven rain is the primary short-to medium-distance dispersal mechanism for CBCD. Long-distance transmission normally occurs from the transportation of infected citrus materials or contaminated equipment. Xac can survive for a long period in field. Though PCR-based methods for diagnosis of CBCD have been described previously, there have some deficiencies in preparation of citrus samples and the specificity a'nd stabilization of detection. Moreover, there is no commercialdiagnosis kit available in China and worldwide. The CBCD quarantine request a new system ofmolecular diagnosis and detection technique with specificity, sensitivity and accuracy characters after China's entry WTO, there has no research report about this field in our country.Objective: This thesis aimed at developing of Xac detection system of DNA molecule with rapid, accuracy, stabilization and practical tool of disease diagnosis for establishment of disease-free area and precautionary monitoring of CBCD through screening of specific primer with detection stabilization and sensitivity, construction of harmless reference system using recombined plasmid and transformed E. coli, modification of sample-preparation method with simpleness and efficiency, and development of diagnosis kit stored at routine temperature.Methods: First of all, primers were designed using Primer Premier 5.0. The specificity had been validated via two ways, one is homologous comparison of target sequence in database of nucleotide sequences, and the other is PCR detection of citrus epiphytes and kindred bacteria of Xac. The sensitivity of PCR was determinated by amplification of .Vac suspension and DNA solution with ten-fold serial dilution. PCR profiles were adjusted by using different type of Thermocycler in different temperature control model at different lab, which ensured the stability of PCR detection. After being inserted into clone vector and recombinant plasmid transformed into the E. coli strain (JM109). the target DNA fragment was sequenced, and amplification fidelity of PCR was verified bysequences alignment. Then a harmless reference system was constructed with recombinant plasmid or bacteria suspension of transformed E. coli, soaking and inspissation procedures were used for varied citrus sample preparation. Solidifying coated PCR reagent kit was produced by addingmacromolecular stabilizer (MS) into PCR mixed reagents and vacuum freeze-drying treatment. Field samples of citrus collected from Sichuan, Guangxi, Hunan provinces and Chongqing area were detected by PCR assay, and some detection results were further confirmed by cultivation and pathogenecity test.Experiment Results:1) Specific primers (JYF5/JYR5) which have good specificity, sensitivity and stability for Xacdetection, were designed based on published sequence encoding a conserved hypothetical proteingene in Xac chromosome. The alignment result of target sequence in database of nucleotide sequences shows that there is no fragment sequence matching with specific primers, and the longest one matched with target fragment is merely 42 bp. PCR amplification product of 413 bp target band of pathogen was generated from each strain of Xac, but not from citrus epiphytes, kindred bacteria of Xac and solution of healthy citrus tissue. Detection sensitivity was 10 cells or 1.56 pg DNA of target bacteria per reaction (25 uL volume). After optimization of amplification condition, stablizing results were obtained by using different type of thermocycler in different temperature control model, but there are a few differences in PCR profiles.2) The sequencing results of cloned target DNA fragment are the same as the reported sequences, and amplification fidelity of PCR was verified. As an important quarantine. pest,...
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